Compositions and methods useful for the regulation of abiotic stress responses in higher plants

ABSTRACT

Compositions and methods for creating plants exhibiting enhanced resistance to abiotic stresses, especially cold stress are disclosed.

This application claims priority to PCT/US2015/034430 filed Jun. 5, 2015which in turn claims priority to U.S. Provisional Application No.62/008,913 filed Jun. 6, 2014, the entire disclosure being incorporatedherein by reference as though set forth in full.

FIELD OF THE INVENTION

This invention relates to the fields of genetic engineering andtransgenic plants. More specifically the invention provides compositionsand methods for regulating ribulose bisphosphate carboxylase/oxygenaseassembly and accumulation in higher plants, thereby altering abioticstress responses and increasing photosynthetic activity in said plant.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the majorenzyme by which green plants, algae, cyanobacteria and other autotrophicorganisms sequester carbon dioxide into organic compounds via theCalvin-Benson pathway (Andersson and Backlund, 2008). Rubisco catalyzesthe photosynthetic carbon reduction and the photorespiratory carbonoxidation reactions of the substrate ribulose-1,5-bisphosphate (RuBP)with CO₂ and O₂, respectively. The inefficiency of Rubisco in fixing CO₂has a limiting impact on agricultural productivity and in compensation,Rubisco accounts for as much as 20-30% and 4-9% of total nitrogencompounds in C₃ and C₄ higher plants, respectively (Feller et al.,2008).

Attempts to improve the catalytic properties of higher plant Rubisco(reviewed in Parry et al., 2003; Mueller-Cajar and Whitney, 2008B) havemet with only modest success, which can be traced in part to the lack ofa comprehensive knowledge of its biogenesis and the absence of an invitro reconstitution system. Form I Rubisco, found in higher plants,algae and cyanobacteria, is a hexadecamer composed of eight large(50-kDa) and eight small (13-15 kDa) subunits, denoted here as LS andSS, respectively. The genes encoding LS (rbcL) and SS (RBCS) are locatedin the chloroplast and nuclear genomes, respectively. SS is expressed asa pre-protein that is translocated into the chloroplast, where itssignal peptide is cleaved prior to its assembly with LS (Nishimura etal., 2008). The two subunits accumulate stoichiometrically in thechloroplast, a phenomenon which is mediated by feedback inhibition of LSsynthesis by unassembled subunits (Rodermel et al., 1996; Wostrikoff andStern, 2007), as well as proteolysis of unassembled SS (Kanevski andMaliga, 1994).

Attempts to delineate the assembly pathway of Form I Rubisco haveexploited two major approaches; in vivo assembly of cyanobacterialRubisco mainly using E. coli cells, and in vitro reconstitution of theenzyme via addition of individual components. In the first approach,assembly of Synechococcus PCC 6301 Rubisco in E. coli resulted in afunctional enzyme (van der Vies et al., 1986; Tabita, 1999). LS was alsoexpressed alone in this way, and shown to have minimal catalyticactivity in the octamer form, which could be enhanced by the subsequentaddition of SS (Andrews, 1988).

Rubisco assembly requires multiple chaperones. The probable role ofchaperonin (Cpn) 60 was first discovered through the co-purification ofchloroplast Rubisco with a protein homologous to E. coli GroEL(Barraclough and Ellis, 1980). It was subsequently demonstrated thatoverexpression of E. coli GroEL-ES significantly promoted the assemblyand activity of Synechococcus Rubisco in E. coli (Goloubinoff et al.,1989a). In fact, E. coli GroEL-ES and Mg-ATP proved to be the onlyfactors necessary for the reconstitution of a catalytically activeRhodospirillum rubrum Form II Rubisco (Goloubinoff et al., 1989b).However, only recently was Form I Rubisco assembled in vitro (Liu etal., 2010), which required both GroEL-ES and a small chaperone calledRbcX (Larimer and Soper, 1993). RbcX appears to play a pivotal role inthe solubility of LS and in vivo assembly of active holoenzyme inSynechococcus strains where the gene is located within the Rubiscooperon (Onizuka et al., 2004; Emlyn-Jones et al., 2006; Saschenbreckeret al., 2007). In maize, the rbcX gene is expressed in leaves (Li etal., 2010), however the polypeptide remains to be detected in proteomicstudies (Friso et al., 2010). Other than Cpn60, the only chloroplastprotein shown to play a direct role in the folding or assembly of plantRubisco is Bundle Sheath Defective 2 (BSD2), a DnaJ-like chaperone (Rothet al., 1996; Brutnell et al., 1999) with an unidentified mechanism ofaction.

Although plant and cyanobacterial Rubisco are both Form I, and theconstituent proteins share over 80% amino acid identity (Parry et al.,2003), higher plant Rubisco has proven refractory to manipulation. Whenexpressed in E. coli, higher plant SS and LS are insoluble, do notassociate with one another to form oligomers, and no enzymatic activityis detectable (Gatenby et al., 1981; Gatenby, 1984; Gatenby et al.,1987; Parry et al., 2003). This implicates additional, and possiblyplant-specific proteins, in higher plant Rubisco biogenesis.

SUMMARY OF THE INVENTION

In accordance with the present invention, modified higher plants areprovided which comprise elevated levels of at least rubisco smallsubunit protein (SS) and ribulose-1,5-BisPhosphate Carboxylase/OxygenaseAccumulation Factor1 (RAF1) when compared to wild type plants. In analternative embodiment, the plants of the invention comprise elevatedlevels of SS, RAF1 and LS. In a particularly preferred embodiment, theplants comprise elevated levels of SS, LS, RAF1 and BSD2 when comparedto wild type. Surprisingly, the present inventors have discovered thatplants comprising elevated levels of these proteins exhibit increasedrubisco content and enhanced resistance to abiotic stress when comparedto plants comprising levels found in untreated or wild type plants. In apreferred embodiment, the plant is maize and the abiotic stress is coldstress. In yet another preferred embodiment, the plants exhibitincreased photosynthetic activity and increased biomass.

In another aspect of the invention, transgenic plants are provided whichcomprise heterologous nucleic acids encoding at least SS and RAF1,thereby producing plants harboring elevated levels of these proteins ascompared to wild type. In another embodiment, the plants compriseheterologous nucleic acids encoding SS, RAF1 and LS. In yet anotherembodiment, the plants comprise heterologous nucleic acids encoding SS,RAF1, LS, and BSD2. These plants also exhibit increased cold resistancewhen compared to wild type plants lacking said heterologous nucleicacids. In certain embodiments, these plants also exhibit increasedphotosynthetic activity and increased biomass.

Also encompassed by the present invention are progeny, seeds and plantcells obtained from the modified or transgenic plants described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Simplified model of the proteins required for Rubisco assemblyin maize.

FIGS. 2A-2D show the constructs used to stably transform maize lines.Arrows represent the maize ubiquitin promoter. Verified lines werecrossed in combinations to create stacked transgenic linesoverexpressing different Rubisco subunits and assembly proteins. LSNdenotes nucleus encoded LS. FIG. 2A: UBISS-LS_(N); FIG. 2B: UBIBSD2;FIG. 2C: UBIRAF1; FIG. 2D: UBISS.

FIG. 3 . Total protein extracted under native conditions on an equalleaf area basis and separated on native 4%-16% gradient acrylamide gels.Gels were stained with coomassie blue, or transferred to PVDF membraneand probed with anti-LS or anti-FLAG antibodies.

FIG. 4 . Total proteins analyzed by immunoblot after Mesophyll (M) andBundle Sheath (BS) cell isolation. A coomassie stained gel is present toreflect loading.

FIGS. 5A-5B (FIG. 5A) Total leaf soluble proteins were extracted on anequal leaf area basis and analyzed by immunoblot. (FIG. 5B) LS abundancewas quantified using the Odyssey scanner and normalized to the PSIIprotein D1 (n=7). Error bars represent the standard deviation; *P<0.0005.

FIG. 6 . Phenotype of transgenic maize leaves under cold stress. At day18 after sowing we introduced chilling temperatures (14° C. day/12° C.night) for two weeks. Photograph shows maize leaves before and afterplants were exposed to 14° C. days/12° C. nights for 0, 1 or 2 weeks.

FIG. 7 . Growth of transgenic maize plants under cold stress. Pictureshowing size difference between 32 day old plants containing differenttransgenes after 14 days in chilling conditions (14° C. days/12° C.nights).

FIG. 8 . Immunoblot analysis of LS before and after exposure to 14° C.day/12° C. night conditions for 2 weeks. Total leaf protein wasextracted on an equal leaf area basis and samples were taken from themid-point of the youngest fully expanded leaf (with leaf collar).Coomassie staining was included to reflect loading.

FIG. 9 . A graph showing increased CO₂ assimilation in lines with higherRubisco content at 14° C.

FIG. 10 . A graph showing excess carbon fixed appears to be incorporatedinto biomass during chilling stress.

DETAILED DESCRIPTION OF THE INVENTION

Most life is ultimately sustained by photosynthesis and its ratelimiting carbon fixing enzyme, Ribulose 1,5-bisphosphatecarboxylase/oxygenase (Rubisco). This enzyme incorporates CO₂ into plantcarbohydrates during photosynthesis. Atmospheric oxygen competes withCO₂ as a substrate for Rubisco, giving rise to photorespiration andmaking Rubisco the rate-limiting step in photosynthesis under certainconditions. Although the relatively simple cyanobacterial Rubisco isamenable to in vitro assembly, the hexadecameric higher plant enzyme hasbeen refractory to such manipulation, due to poor understanding of itsassembly pathway.

In accordance with the present invention, we created transgenic maizelines with enhanced Rubisco abundance. The data presented herein showthat overexpression of Rubisco assembly factors and subunits will confera physiological advantage to maize, specifically under cold stressconditions where Rubisco activity is limiting. Rubisco content haspreviously been increased 24-30% in transgenic rice, on a leaf areabasis, through overexpression of endogenous SS (Suzuki et al. 2007) or asorghum SS gene (Ishikawa et al. 2011). In both cases Rubisco activitywas slightly decreased and the photosynthetic rate was not improved,even when Rubisco kcat was significantly increased. In maize, wepreviously overexpressed a nucleus-transplanted copy of the LS gene(LSN), and SS, both under control of the ubiquitin promoter (UBI-LS-SS).While both transgenes were expressed at a high level and nucleus-encodedLS was readily incorporated into holoenzyme, no overall change inRubisco abundance was observed (Wostrikoff et al. 2012). Thus, in C3rice increasing Rubisco abundance did not confer a photosyntheticadvantage, and in C4 maize simply increasing subunit expression did notaffect holoenzyme accumulation. From genetic analysis, we now know thatapart from LS and SS, BSD2, RAF1, CPS2/Cpn60 and RAF2 are all requiredfor Rubisco assembly and/or stability in maize. Taking advantage of thisknowledge we have added ubiquitin promoter-driven expression of RAF1, orboth BSD2 and RAF1 to the UBI-LS-SS line, which as shown below increasesLS, and presumably holoenzyme abundance. Ubi-RAF2 transgenics can alsobe generated and can be introgressed into this background for apotential incremental increase above what we have observed.

The following definitions are provided to facilitate an understanding ofthe present invention.

As used herein, “genetically altered” means the modified expression ofat least two, three or four of RAF1, a BSD2, LS, SS protein or both, orfunctional mutants thereof resulting from one or more geneticmodifications; the modifications including but not limited to:recombinant gene technologies, induced mutations, and breeding stablygenetically modified plants to produce progeny and seed comprising thealtered gene product.

A “modified plant” is a plant that has been treated with an agent thatincreases expression of endogenous levels of at least SS/RAF1 whencompared to wild type untreated plants. Alternatively the plant may betreated with an agent that elevates expression of SS/RAF1 and LS. In yetanother embodiment, the treatment results in elevated levels ofSS/RAF1/LS and BSD2.

A “transgenic plant” refers to a plant whose genome has been altered bythe introduction of at least one heterologous nucleic acid molecule.Transgenic plants comprising altered RAF1 protein are provided herein.

The term “decreased” is intended to mean that the measurement of aparameter is changed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200% or more when compared to the measurement of thatparameter in a suitable control.

The term “increased” is intended to mean that the measurement of aparameter is changed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200% or more when compared to the measurement of thatparameter in a suitable control.

The terms “inhibit,” “inhibition,” “inhibiting”, “reduced”, “reduction”and the like as used herein refer to any decrease in the expression orfunction of a target gene product, including any relative decrement inexpression or function up to and including complete abrogation ofexpression or function of the target gene product.

The terms “promote,” “upregulate,” “increase”, and “overexpress”, andthe like as used herein refer to any increase in the expression orfunction of a target gene product, including any relative increase inexpression or function of the target gene product.

The term “expression” as used herein in the context of a gene productrefers to the biosynthesis of that gene product, including thetranscription and/or translation of the gene product. Inhibition ofexpression or function of a target gene product (i.e., a gene product ofinterest) can be in the context of a comparison between any two plants,for example, expression or function of a target gene product (e.g.,protein) in a genetically altered plant versus the expression orfunction of that target gene product in a corresponding wild-type plant.Expression levels can also be used to refer to increases or decreases inprotein levels due to alterations in stability or assembly of suchproteins. Inhibition of expression or function of the target geneproduct can be in the context of a comparison between plant cells,organelles, organs, tissues, or plant parts within the same plant orbetween plants, and includes comparisons between developmental ortemporal stages within the same plant or between plants. Any method orcomposition that down-regulates expression of a target gene product,either at the level of transcription, translation, or stability ordown-regulates functional activity of the target gene product can beused to achieve inhibition of expression or function of the target geneproduct.

The term “inhibitory sequence” encompasses any polynucleotide orpolypeptide sequence that is capable of inhibiting the expression of atarget gene product, for example, at the level of transcription ortranslation, or which is capable of inhibiting the function of a targetgene product. Exemplary constructs encoding such inhibitory sequencesare disclosed herein.

When the phrase “capable of inhibiting” is used in the context of apolynucleotide inhibitory sequence, it is intended to mean that theinhibitory sequence itself exerts the inhibitory effect; or, where theinhibitory sequence encodes an inhibitory nucleotide molecule (forexample, hairpin RNA, miRNA, or double-stranded RNA polynucleotides), orencodes an inhibitory polypeptide (i.e., a polypeptide that inhibitsexpression or function of the target gene product), following itstranscription (for example, in the case of an inhibitory sequenceencoding a hairpin RNA, miRNA, or double-stranded RNA polynucleotide) orits transcription and translation (in the case of an inhibitory sequenceencoding an inhibitory polypeptide), the transcribed or translatedproduct, respectively, exerts the inhibitory effect on the target geneproduct (i.e., inhibits expression or function of the target geneproduct).

Conversely, the terms “increase”, “increased”, and “increasing” in thecontext of the methods of the present invention refer to any increase inthe expression or function of a gene product, including any relativeincrement in expression or function.

In many instances the nucleotide sequences for use in the methods of thepresent invention, are provided in transcriptional units fortranscription in the plant of interest. A transcriptional unit iscomprised generally of a promoter and a nucleotide sequence operablylinked in the 3′ direction of the promoter, optionally with aterminator.

“Operably linked” refers to the functional linkage between a promoterand a second sequence, wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondsequence. The expression cassette will include 5′ and 3′ regulatorysequences operably linked to at least one of the sequences of theinvention.

Generally, in the context of an over expression cassette, operablylinked means that the nucleotide sequences being linked are contiguousand, where necessary to join two or more protein coding regions,contiguous and in the same reading frame. In the case where anexpression cassette contains two or more protein coding regions joinedin a contiguous manner in the same reading frame, the encodedpolypeptide is herein defined as a “heterologous polypeptide” or a“chimeric polypeptide” or a “fusion polypeptide”. The cassette mayadditionally contain at least one additional coding sequence to beco-transformed into the organism. Alternatively, the additional codingsequence(s) can be provided on multiple expression cassettes.

With regard to nucleic acids used in the invention, the term “isolatednucleic acid” is sometimes employed. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated nucleic acid” may comprise a DNA molecule inserted into avector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryote or eukaryote. An “isolated nucleic acidmolecule” may also comprise a cDNA molecule. An isolated nucleic acidmolecule inserted into a vector is also sometimes referred to herein asa recombinant nucleic acid molecule.

With respect to RNA molecules, the term “isolated nucleic acid”primarily refers to an RNA molecule encoded by an isolated DNA moleculeas defined above. Alternatively, the term may refer to an RNA moleculethat has been sufficiently separated from RNA molecules with which itwould be associated in its natural state (i.e., in cells or tissues),such that it exists in a “substantially pure” form.

By the use of the term “enriched” in reference to nucleic acid it ismeant that the specific DNA or RNA sequence constitutes a significantlyhigher fraction (2-5 fold) of the total DNA or RNA present in the cellsor solution of interest than in normal cells or in the cells from whichthe sequence was taken. This could be caused by a person by preferentialreduction in the amount of other DNA or RNA present, or by apreferential increase in the amount of the specific DNA or RNA sequence,or by a combination of the two. However, it should be noted that“enriched” does not imply that there are no other DNA or RNA sequencespresent, just that the relative amount of the sequence of interest hasbeen significantly increased.

It is also advantageous for some purposes that a nucleotide sequence bein purified form. The term “purified” in reference to nucleic acid doesnot require absolute purity (such as a homogeneous preparation);instead, it represents an indication that the sequence is relativelypurer than in the natural environment (compared to the natural level,this level should be at least 2-5 fold greater, e.g., in terms ofmg/ml). Individual clones isolated from a cDNA library may be purifiedto electrophoretic homogeneity. The claimed DNA molecules obtained fromthese clones can be obtained directly from total DNA or from total RNA.The cDNA clones are not naturally occurring, but rather are preferablyobtained via manipulation of a partially purified naturally occurringsubstance (messenger RNA). The construction of a cDNA library from mRNAinvolves the creation of a synthetic substance (cDNA) and pureindividual cDNA clones can be isolated from the synthetic library byclonal selection of the cells carrying the cDNA library. Thus, theprocess, which includes the construction of a cDNA library from mRNA andisolation of distinct cDNA clones, yields an approximately 10⁻⁶-foldpurification of the native message. Thus, purification of at least oneorder of magnitude, preferably two or three orders, and more preferablyfour or five orders of magnitude is expressly contemplated. Thus theterm “substantially pure” refers to a preparation comprising at least50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, etc.). More preferably, the preparation comprises atleast 75% by weight, and most preferably 90-99% by weight, the compoundof interest. Purity is measured by methods appropriate for the compoundof interest.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be complementary to different strands of aparticular target nucleic acid sequence. This means that the probes mustbe sufficiently complementary so as to be able to “specificallyhybridize” or anneal with their respective target strands under a set ofpre-determined conditions. Therefore, the probe sequence need notreflect the exact complementary sequence of the target. For example, anon-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarity with the sequenceof the target nucleic acid to anneal therewith specifically.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos.4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which areincorporated by reference herein.

The term “vector” relates to a single or double stranded circularnucleic acid molecule that can be infected, transfected or transformedinto cells and replicate independently or within the host cell genome. Acircular double stranded nucleic acid molecule can be cut and therebylinearized upon treatment with restriction enzymes. An assortment ofvectors, restriction enzymes, and the knowledge of the nucleotidesequences that are targeted by restriction enzymes are readily availableto those skilled in the art, and include any replicon, such as aplasmid, cosmid, bacmid, phage or virus, to which another geneticsequence or element (either DNA or RNA) may be attached so as to bringabout the replication of the attached sequence or element. A nucleicacid molecule of the invention can be inserted into a vector by cuttingthe vector with restriction enzymes and ligating the two piecestogether.

Many techniques are available to those skilled in the art to facilitatetransformation, transfection, or transduction of the expressionconstruct into a prokaryotic or eukaryotic organism. The terms“transformation”, “transfection”, and “transduction” refer to methods ofinserting a nucleic acid and/or expression construct into a cell or hostorganism. These methods involve a variety of techniques, such astreating the cells with high concentrations of salt, an electric field,or detergent, to render the host cell outer membrane or wall permeableto nucleic acid molecules of interest, microinjection, PEG-fusion, andthe like.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA.

In one embodiment, the promoter element of the present inventionprecedes the 5′ end of the recombinant nucleic acid molecule such thatthe latter is transcribed into mRNA. Host cell machinery then translatesmRNA into a polypeptide.

Those skilled in the art will recognize that a nucleic acid vector cancontain nucleic acid elements other than the promoter element and theRAF1 and or BSD2 coding nucleic acid molecule. These other nucleic acidelements include, but are not limited to, origins of replication,ribosomal binding sites, nucleic acid sequences encoding drug resistanceenzymes or amino acid metabolic enzymes, and nucleic acid sequencesencoding secretion signals, localization signals, or signals useful forpolypeptide purification.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, plastid, phage or virus that is capable of replication largelyunder its own control. A replicon may be either RNA or DNA and may besingle or double stranded.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is readily measurable,e.g., by biological assay, immunoassay, radio immunoassay, or bycolorimetric, fluorogenic, chemiluminescent or other methods. GFP isexemplified herein. The nucleic acid may be either RNA or DNA, linear orcircular, single or double stranded, and is operatively linked to thenecessary control elements for the expression of the reporter geneproduct. The required control elements will vary according to the natureof the reporter system and whether the reporter gene is in the form ofDNA or RNA, but may include, but not be limited to, such elements aspromoters, enhancers, translational control sequences, poly A additionsignals, transcriptional-termination signals and the like.

The term “selectable marker gene” refers to a gene that when expressedconfers a selectable phenotype, such as herbicide tolerance, on atransformed plant cell.

The terms “recombinant plant,” or “transgenic plant” refer to plants,which have a new combination of genes or nucleic acid molecules. A newcombination of genes or nucleic acid molecules can be introduced into aplant using a wide array of nucleic acid manipulation techniquesavailable to those skilled in the art.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins, with which it wouldnaturally be associated, so as to exist in “substantially pure” form.“Isolated” is not meant to exclude artificial or synthetic mixtures withother compounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

A “specific binding pair” comprises a specific binding member (sbm) anda binding partner (bp), which have a particular specificity for eachother and which in normal conditions bind to each other in preference toother molecules. Examples of specific binding pairs are antigens andantibodies, ligands and receptors and complementary nucleotidesequences. The skilled person is aware of many other examples. Further,the term “specific binding pair” is also applicable where either or bothof the specific binding member and the binding partner comprise a partof a large molecule. In embodiments in which the specific binding paircomprises nucleic acid sequences, they will be of a length to hybridizeto each other under conditions of the assay, preferably greater than 10nucleotides long, more preferably greater than 15 or 20 nucleotideslong.

Preparation of SS, RAF1, LS, and BSD2 Encoding Nucleic Acid Moleculesand Creation of Transgenic Plants Containing the Same

Nucleic acid molecules of the invention encoding desired polypeptidesmay be prepared by two general methods: (1) synthesis from appropriatenucleotide triphosphates, or (2) isolation from biological sources. Bothmethods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as the DNAsequences encoding LS, SS, RAF1 and/or BSD2, enables preparation of anisolated nucleic acid molecule of the invention by oligonucleotidesynthesis. Synthetic oligonucleotides may be prepared by thephosphoramidite method employed in the Applied Biosystems 38A DNASynthesizer or similar devices. The resultant construct may be useddirectly or purified according to methods known in the art, such as highperformance liquid chromatography (HPLC).

Specific probes/primers for identifying such sequences as the LS, SS,RAF1 or BSD2 encoding sequence may be between 15 and 40 nucleotides inlength. For probes/primers longer than those described above, theadditional contiguous nucleotides are provided the nucleic acidsequences in GenBank for these proteins.

Additionally, cDNA or genomic clones having homology with LS, SS, RAF1and/or BSD2 may be isolated from other species using oligonucleotideprobes corresponding to predetermined sequences within the targetnucleic acids of the invention. Alternatively, the cDNA may be amplifiedby reverse transcriptase after making cDNA from the pool. The sequencedmaize genome database provides the full length of cDNA and CDS. Suchhomologous sequences encoding the proteins of intereset may beidentified by using hybridization and washing conditions of appropriatestringency. For example, hybridizations may be performed, according tothe method of Sambrook et al., Molecular Cloning, Cold Spring HarborLaboratory (1989), using a hybridization solution comprising: 5×SSC,5×Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmonsperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.Hybridization is carried out at 37-42° C. for at least six hours.Following hybridization, filters are washed as follows: (1) 5 minutes atroom temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperaturein 2×SSC and 0.1% SDS; (3) 30 minutes 1 hour at 37° C. in 1×SSC and 1%SDS; (4) 2 hours at 42-65° C. in 1×SSC and 1% SDS, changing the solutionevery 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules of a specifiedsequence homology (Sambrook et al., 1989) is as follows: T_(m)81.5°C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp in duplex.

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 11.5°C. with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated T_(m) of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12-20° C. below the T_(m) of the hybrid. In regards tothe nucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

Also encompassed within the scope of the invention are transgenic plantscontaining the aforementioned RAF1- and/or BSD2 encoding nucleic acids,or fragments or derivatives thereof.

The following example is provided to illustrate certain embodiments ofthe invention. It is not intended to limit the invention in any way.

Example I Transgenic Plant Performance Under Chilling Temperatures

Due to the strong limitation Rubisco imposes on CO₂ assimilation at lowtemperatures, increasing Rubisco content may be a solution to enhanceacclimation to cold in maize, as acclimation involving changes inRubisco content does occur in some cool tolerant C4 species, such asMiscanthus (Dwyer et al. 2007, Sage and Kubien 2007). To our knowledge,this has never been directly been tested. We show that by overexpressionof Rubisco produces high enough concentrations to avoid being limitingat low temperatures. The maize ubiquitin promoter was capable ofoverexpressing cold tolerance genes in rice under stress conditions (Itoet al. 2006), and should therefore be equally effective in maize grownat 14° C. Additionally, Rubisco activase, Cpn60, LS and SS maintain highaverage mRNA expression during chilling (Spence 2012). This suggeststhat decreases in Rubisco content in response to chilling occurpost-transcriptionally or post-translationally, i.e. at thetranslational or protein stability level. Thus in principle, byincreasing Rubisco content at least 40% we should be able to compensatefor the ˜40% decrease in Rubisco accumulation seen in maize underchilling temperatures.

Chloroplast-encoded LS interacts with the chaperonin complex tocorrectly fold the newly synthesized protein (Native LS).Nucleus-encoded SS is refolded after being translocated from the cytosolvia the Tic-Toc complex. The data presented here show that severalproteins, RAF2, RAF1 and/or BSD2 are involved in refolding imported SS,and also in forming assembly intermediates that capture folded LS oncereleased from the chaperonin complex. In the absence of RAF2, RAF1 orBSD2, LS is subject to aggregation and proteolysis. Marginal holoenzymeassembly does occur in the absence of RAF2, thus it is possible that itsrole can be bypassed by RAF1 and BSD2. A schematic drawing of thiscomplex is shown in FIG. 1 .

We employed a transgenic approach to highly express Rubisco subunits andassembly proteins. See FIG. 2 wherein combinations of constructs A+B,C+D, A+C, and A+B+C were created. The results shown in FIGS. 3 and 4demonstrate that Rubisco is assembled as the holoenzyme in bundle sheathcells. Total rubisco migrated at 550 KD, indicating that Rubisco issoluble and assembled into the hexadecameric form. PEPC (present in Mcells only) and ME (only present in BS cells) were used to evaluate cellseparation purity. From this, LS appears to be accumulating only in BScells.

To see if there was an obvious whole plant phenotype, we germinatedUBI-RAF1, UBI-RAF1-SS, UBI-BSD2-LSN-SS-RAF1 and WT plants under 25° C.day/20° C. night conditions under high light (500 μmol photons m-2s-1).At day 18 after sowing we introduced chilling temperatures (14° C.day/12° C. night) for two weeks. 14° C. was used because maize has beenshown to lose a significant proportion of its ability to assimilate CO₂at and below that temperature (Naidu and Long 2004). We observed that asignificant LS increase was found in the UBI-RAF1-SS (C+D) and theUBI-BSD2-LS_(N)-SS-RAF1 (A+B+C) lines (FIG. 5 ). FIG. 6 shows leavesbefore and after plants were exposed to 14° C. days/12° C. nights for 0,1 or 2 weeks. The WT in this experiment was inbred B73, which isslightly less vigorous than the transgenic lines, which were created inthe Hi-II hybrid background. Irrespective of size, the WT and UBI-RAF1became increasingly chlorotic, whereas UBI-RAF1-SS andUBI-BSD2-LSN-SS-RAF1 largely maintained their chlorophyll content. Atthe end of the experiment (FIG. 7 ), WT and UBI-RAF1 plants appearedboth chlorotic and stunted compared to the lines which had been found toharbor increased Rubisco at normal growth temperatures (FIG. 5 ). Thecomparison between UBI-RAF1 and UBI-RAF1-SS/UBIBSD2-LS_(N)-SS-RAF1 isparticularly informative, because all are Hi-II derivatives propagatedby selfing or outcrossing to other Hi-II lines, and their statures weresimilar at the outset of the experiment.

LS accumulation was measured in plants pre- and post cold stress, asshown in FIG. 8 . We found that in the WT control, Rubisco decreased˜40% at chilling temperatures, in agreement with previous observations(Naidu et al. 2003, Spence 2012, Wang et al. 2008a). On the other hand,the decrease in Rubisco was mitigated in both UBI-RAF1-SS andUBI-BSD2-LS_(N)-SSRAF1 transgenic plants. Quantification of the gels inFIG. 8 indicates that these lines have comparable levels of Rubiscoaccumulation in the cold, to what WT plants possess at optimal growthtemperatures. Thus, the apparent tolerance to chilling can be correlatedwith maintenance of sufficient Rubisco content. This correlation of highexpression of Rubisco LS protein with maize tolerance to chillingtemperatures provides an indication that the transgenic plants describedare resistant to other abiotic stresses and provides the means to createsuch plants.

Gas exchange can be used to measures the rate of photosynthetic carbonassimilation. We performed additional studies to ascertain whethermaintenance of rubisco content at low temperatures is important forsustaining photosynthetic capacity. FIG. 9 shows that the transgenicplants of the invention with higher rubisco content show increased CO₂assimilation at 14° C. FIG. 10 shows that excess carbon fixed appears tobe incorporated into biomass during chilling stress.

The results presented herein show that rubisco content can be increasedby overexpression of the subunits and assembly factors and that theoverexpressed rubisco was present as holoenzyme in bundle sheathchloroplasts. Increased Rubisco content correlates with much higherphotosynthetic rates and increased biomass and leaf area under chillingconditions. Notably, increasing Rubisco appears to lessen the effects ofchilling stress in higher plants, and in maize in particular.

REFERENCES

-   (1) Long, S. P. (1983) Plant Cell Environ., 6, 345-363.-   (2) Wang, D., Portis, A. R., Jr., Moose, S. P. and    Long, S. P. (2008) Plant Physiol., 148, 557-567.-   (3) Naidu, S. L. and Long, S. P. (2004) Planta, 220, 145-155.-   (4) Naidu, S. L., Moose, S. P., AK, A. L.-S., Raines, C. A. and    Long, S. P. (2003) Plant Physiol., 132, 1688-169-   (5) Spence, A. K. (2012) Plant Biology. Urbana, Illinois: Univ.    Illinois Urbana-Champaign, pp. 146.-   (6) Suzuki et al. (2007) Plant Cell Physiol., 48:626-637.-   (7) Whitney et al. (2015) Proc Natl Acad Sci USA. 112: 3564-3569.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope of the presentinvention, as set forth in the following claims.

What is claimed is:
 1. A transgenic C4 plant comprising heterologous, recombinant nucleic acids for over-expressing rubisco small subunit (SS), rubisco large subunit (LS), and ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 (RAF1), over expression of said SS, LS and RAF1 increasing holoenzyme accumulation, said plant exhibiting increased rubisco content, increased photosynthetic rate, increased biomass and enhanced resistance to abiotic stress when compared to plants lacking said heterologous nucleic acids.
 2. The transgenic plant of claim 1, which is a maize plant.
 3. The transgenic plant of claim 1, further comprising a heterologous nucleic acid encoding bundle sheath defective 2 (BSD2).
 4. The transgenic plant of claim 1, wherein said abiotic stress is cold stress.
 5. The transgenic plant of claim 1, wherein said RAF1, SS and LS are operably linked to a ubiquitin promoter.
 6. The transgenic plant of claim 1, wherein said RAF1, SS and LS heterologous, recombinant nucleic acids are from the same species of plant to be transformed. 